The present invention relates generally to methods for modulating endothelial cell adhesion, and more particularly to cyclic peptides comprising a cadherin cell adhesion recognition sequence, and to the use of such cyclic peptides for inhibiting or enhancing cadherin-mediated endothelial cell functions, such as cell adhesion.
Cell adhesion is a complex process that is important for maintaining tissue integrity and generating physical and permeability barriers within the body. All tissues are divided into discrete compartments, each of which is composed of a specific cell type that adheres to similar cell types. Such adhesion triggers the formation of intercellular junctions (i.e., readily definable contact sites on the surfaces of adjacent cells that are adhering to one another), also known as tight junctions, gap junctions and belt desmosomes. The formation of such junctions gives rise to physical and permeability barriers that restrict the free passage of cells and other biological substances from one tissue compartment to another. For example, the blood vessels of all tissues are composed of endothelial cells. In order for components in the blood to enter a given tissue compartment, they must first pass from the lumen of a blood vessel through the barrier formed by the endothelial cells of that vessel. Similarly, in order for substances to enter the body via the gut, the substances must first pass through a barrier formed by the epithelial cells of that tissue. To enter the blood via the skin, both epithelial and endothelial cell layers must be crossed.
Cell adhesion is mediated by specific cell surface adhesion molecules (CAMs). There are many different families of CAMs, including the immunoglobulin, integrin, selectin and cadherin superfamilies, and each cell type expresses a unique combination of these molecules. Cadherins are a rapidly expanding family of calcium-dependent CAMs (Munro et al., In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RG Landes Co. (Austin Tex., 1996). The classical cadherins (abbreviated CADs) are integral membrane glycoproteins that generally promote cell adhesion through homophilic interactions (a CAD on the surface of one cell binds to an identical CAD on the surface of another cell), although CADs also appear to be capable of forming heterotypic complexes with one another under certain circumstances and with lower affinity. Cadherins have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different CADs expressed on different cell types. N (neural)xe2x80x94cadherin is predominantly expressed by neural cells, endothelial cells and a variety of cancer cell types. E (epithelial)xe2x80x94cadherin is predominantly expressed by epithelial cells. Other CADs are P (placental)xe2x80x94cadherin, which is found in human skin and R (retinal)xe2x80x94cadherin. A detailed discussion of the classical cadherins is provided in Munro S B et al., 1996, In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp.17-34 (RG Landes Company, Austin Tex.).
The structures of the CADs are generally similar. As illustrated in FIG. 1, CADs are composed of five extracellular domains (EC1-EC5), a single hydrophobic domain (TM) that transverses the plasma membrane (PM), and two cytoplasmic domains (CP1 and CP2). The calcium binding motifs DXNDN (SEQ ID NO:9), DXD and LDRE (SEQ ID NO:9) are interspersed throughout the extracellular domains. The first extracellular domain (EC1) contains the classical cadherin cell adhesion recognition (CAR) sequence, HAV (His-Ala-Val), along with flanking sequences on either side of the CAR sequence that may play a role in conferring specificity. Synthetic peptides containing the CAR sequence and antibodies directed against the CAR sequence have been shown to inhibit CAD-dependent processes (Munro et al., supra; Blaschuk et al., J. Mol. Biol. 211:679-82, 1990; Blaschuk et al., Develop. Biol. 139:227-29, 1990; Alexander et al., J. Cell. Physiol. 156:610-18, 1993). The three-dimensional solution and crystal structures of the EC1 domain have been determined (Overduin et al., Science 267:386-389, 1995; Shapiro et al., Nature 374:327-337, 1995).
Although cell adhesion is required for certain normal physiological functions, there are situations in which cell adhesion is undesirable. For example, many pathologies (such as autoimmune and inflammatory diseases) involve abnormal cellular adhesion. Cell adhesion may also play a role in graft rejection. In such circumstances, modulation of cell adhesion may be desirable.
In addition, permeability barriers arising from cell adhesion create difficulties for the delivery of drugs to specific tissues and tumors within the body. For example, skin patches are a convenient tool for administering drugs through the skin. However, the use of skin patches has been limited to small, hydrophobic molecules because of the epithelial and endothelial cell barriers. Similarly, endothelial cells render the blood capillaries largely impermeable to drugs, and the blood/brain barrier has hampered the targeting of drugs to the central nervous system. In addition, many solid tumors develop internal barriers that limit the delivery of anti-tumor drugs and antibodies to inner cells.
Attempts to facilitate the passage of drugs across such barriers generally rely on specific receptors or carrier proteins that transport molecules across barriers in vivo. However, such methods are often inefficient, due to low endogenous transport rates or to the poor functioning of a carrier protein with drugs. While improved efficiency has been achieved using a variety of chemical agents that disrupt cell adhesion, such agents are typically associated with undesirable side-effects, may require invasive procedures for administration and may result in irreversible effects. It has been suggested that linear synthetic peptides containing a cadherin CAR sequence may be employed for drug transport (WO 91/04745), but such peptides are often metabolically unstable and are generally considered to be poor therapeutic agents.
Accordingly, there is a need in the art for compounds that modulate cell adhesion and improve drug delivery across permeability barriers without such disadvantages. The present invention fulfills this need and further provides other related advantages.
The present invention provides modulating agents comprising cyclic peptides, and methods for using such agents to inhibit or enhance cadherin-mediated endothelial cell adhesion. Such cyclic peptides generally comprise the sequence His-Ala-Val. Within certain aspects, such cyclic peptides have the formula: 
wherein X1, and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein X1 and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within X1 and X2 ranges from 1 to 12; wherein Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2; and wherein Z1 and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds. Such cyclic peptides may comprise modifications such as an N-acetyl or N-alkoxybenzyl group and/or a C-terminal amide or ester group. Cyclic peptides may be cyclized via, for example, a disulfide bond; an amide bond between terminal functional groups, between residue side-chains or between one terminal functional group and one residue side chain; a thioether bond or xcex41xcex41-ditryptophan, or a derivative thereof.
Within certain embodiments, a cyclic peptide has the formula:

wherein Y1 and Y2 are optional and, if present are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein Y1 and Y2 range in size from 0 to 10 residues; and wherein X and Z are independently selected from the group consisting of amino acid residues, wherein a disulfide bond is formed between residues X and Z; and wherein X has a terminal modification (e.g., an N-acetyl group).
Within further embodiments, a cyclic peptide has the formula: 
wherein Z1 and Z2 are selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein Z1 and Z2 range in size from 1 to 10 residues; and wherein X and Y are independently selected from the group consisting of amino acid residues, wherein a disulfide bond is formed between residues X and Y; and wherein X has a terminal modification (e.g., an N-acetyl group).
Certain specific cyclic peptides provided by the present invention include Nxe2x80x94Ac-CHAVC-NH2 (SEQ ID NO:10), Nxe2x80x94Ac-CHAVC-Yxe2x80x94NH2 (SEQ ID NO:10), Nxe2x80x94Ac-YCHAVC-NH2 (SEQ ID NO:54), Nxe2x80x94Ac-CHAVDC-NH2 (SEQ ID NO:20), Nxe2x80x94Ac-CHAVDIC-NH2 (SEQ ID NO:50), Nxe2x80x94Ac-CHAVDINC-NH2 (SEQ ID NO:51, Nxe2x80x94Ac-CHAVDINGC-NH2 (SEQ ID NO:52), Nxe2x80x94Ac-CAHAVC-NH2 (SEQ ID NO:22), Nxe2x80x94Ac-CAHAVDC-NH2 (SEQ ID NO:26), Nxe2x80x94Ac-CAHAVDIC-NH2 (SEQ ID NO:24), Nxe2x80x94Ac-CRAHAVDC-NH2 (SEQ ID NO:28), Nxe2x80x94Ac-CLRAHAVC-NH2 (SEQ ID NO:30), Nxe2x80x94Ac-CLRAHAVDC-NH2 (SEQ ID NO:32), Nxe2x80x94Ac-CSHAVC-NH2 (SEQ ID NO:36), Nxe2x80x94Ac-CFSHAVC-NH2 (SEQ ID NO:85), Nxe2x80x94Ac-CLFSHAVC-NH2 (SEQ ID NO:86), Nxe2x80x94Ac-CHAVSC-NH2 (SEQ ID NO:38), Nxe2x80x94Ac-CSHAVSC-NH2 (SEQ ID NO:40), Nxe2x80x94Ac-CSHAVSSC-NH2 (SEQ ID NO:42), Nxe2x80x94Ac-CHAVSSC-NH2 (SEQ ID NO:44), Nxe2x80x94Ac-KHAVD-NH2 (SEQ ID NO:12), Nxe2x80x94Ac-DHAVK-NH2 (SEQ ID NO:14), Nxe2x80x94Ac-KHAVE-NH2 (SEQ ID NO:16), Nxe2x80x94Ac-AHAVDI-NH2 (SEQ ID NO:34), Nxe2x80x94Ac-SHAVDSS-NH2 (SEQ ID NO:77), Nxe2x80x94Ac-KSHAVSSD-NH2 (SEQ ID NO:48), Nxe2x80x94Ac-CHAVC-S-NH2 (SEQ ID NO:87), Nxe2x80x94Acxe2x80x94S-CHAVC-NH2 (SEQ ID NO:88), Nxe2x80x94Ac-CHAVC-SSxe2x80x94NH2 (SEQ ID NO:89), Nxe2x80x94Acxe2x80x94S-CHAVC-Sxe2x80x94NH2 (SEQ ID NO:90), Nxe2x80x94Ac-CHAVC-Txe2x80x94NH2 (SEQ ID NO:91), Nxe2x80x94Ac-CHAVC-Exe2x80x94NH2 (SEQ ID NO:92), Nxe2x80x94Ac-CHAVC-Dxe2x80x94NH2 (SEQ ID NO:93), Nxe2x80x94Ac-CHAVYC-NH2 (SEQ ID NO:94), CH3xe2x80x94SO2xe2x80x94HN-CHAVC-Yxe2x80x94NH2 (SEQ ID NO:95), CH3xe2x80x94SO2xe2x80x94HN-CHAVC-NH2 (SEQ ID NO:96), HC(O)xe2x80x94NH-CHAVC-NH2 (SEQ ID NO:96), Nxe2x80x94Ac-CHAVPen-NH2 (SEQ ID NO:79), Nxe2x80x94Ac-PenHAVC-NH2 (SEQ ID NO:80) and Nxe2x80x94Ac-CHAVPC-NH2 (SEQ ID NO:81), as well as derivatives thereof in which the Nxe2x80x94Ac group is replaced by a different terminal group. Within further aspects, the present invention provides cell adhesion modulating agents that comprise a cyclic peptide as described above. Within specific embodiments, such modulating agents may be linked to one or more of a targeting agent, a drug, a solid support or support molecule, or a detectable marker. In addition, or alternatively, a cell adhesion modulating agent may further comprising one or more of: (a) a cell adhesion recognition sequence that is bound by an adhesion molecule other than a cadherin, wherein the cell adhesion recognition sequence is separated from any HAV sequence(s) by a linker; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than a cadherin.
The present invention further provides pharmaceutical compositions comprising a cell adhesion modulating agent as described above, in combination with a pharmaceutically acceptable carrier. Such compositions may further comprise a drug. Alternatively, or in addition, such compositions may comprise: (a) a peptide comprising a cell adhesion recognition sequence that is bound by an adhesion molecule other than a cadherin; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than a cadherin.
Within further aspects, methods are provided for modulating endothelial cell adhesion, comprising contacting a cadherin-expressing endothelial cell with a cell adhesion modulating agent as described above. In certain such aspects, the agent inhibits N-cadherin mediated cell adhesion, resulting in the reduction of unwanted endothelial cell adhesion in the mammal.
The present invention also provides, within other aspects, methods for inhibiting angiogenesis in a mammal, comprising administering to a mammal a cell adhesion modulating agent as described above, wherein the modulating agent inhibits endothelial cell adhesion.
Within further aspects, methods are provided for increasing vasopermeability in a mammal, comprising administering to a mammal a cell adhesion modulating agent as described above, wherein the modulating agent inhibits endothelial cell adhesion.
The present invention further provides, within other aspects, methods for increasing blood flow to a tumor, comprising contacting a tumor with a modulating agent as described above, wherein the modulating agent inhibits endothelial cell adhesion.
Methods are also provided, within further aspects, for disrupting neovasculature in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the modulating agent inhibits endothelial cell adhesion.
Within further aspects, methods are provided for inhibiting the development of endometriosis in a mammal, comprising administering to a mammal a modulating agent as described above, wherein the agent inhibits endothelial cell adhesion.